Effect of aging on the formation and growth of colonic epithelial organoids by changes in cell cycle arrest through TGF-ß-Smad3 signaling.

BACKGROUND: This study aimed to investigate how aging alters the homeostasis of the colonic intestinal epithelium and regeneration after tissue injury using organoid models and to identify its underlying molecular mechanism. METHODS: To investigate aging-related changes in the colonic intestinal epithelium, we conducted organoid cultures from old (older than 80 weeks) and young (6-10 weeks) mice and compared the number and size of organoids at day 5 of passage 0 and the growth rate of organoids between the two groups. RESULTS: The number and size of organoids from old mice was significantly lower than that from young mice (p < 0.0001) at day 5 of passage 0. The growth rate of old-mouse organoids from day 4 to 5 of passage 0 was significantly slower than that of young-mouse organoids (2.21 times vs. 1.16 times, p < 0.001). RNA sequencing showed that TGF-ß- and cell cycle-associated genes were associated with the aging effect. With regard to mRNA and protein levels, Smad3 and p-Smad3 in the old-mouse organoids were markedly increased compared with those in the young-mouse organoids. Decreased expression of ID1, increased expression of p16INK4a, and increased cell cycle arrest were observed in the old mouse-organoids. Treatment with SB431542, a type I TGF-ß receptor inhibitor, significantly increased the formation and growth of old-mouse organoids, and TGF-ß1 treatment markedly suppressed the formation of young-mouse organoids. In the acute dextran sulfate sodium-colitis model and its organoid experiments, the colonic epithelial regeneration after tissue injury in old mice was significantly decreased compared with young mice. CONCLUSIONS: Aging reduced the formation ability and growth rate of colonic epithelial organoids by increasing cell cycle arrest through TGF-ß-Smad3-p16INK4a signaling.

Inheritance of Charcot-Marie-Tooth disease 1A with rare nonrecurrent genomic rearrangement.

Rare copy number variations by the nonrecurrent rearrangements involving PMP22 have been recently suggested to be associated with CMT1A peripheral neuropathy. As a mechanism of the nonrecurrent rearrangement, replication-based fork stalling template switching (FoSTeS) by microhomology-mediated break-induced replication (MMBIR) has been proposed. We found three Korean CMT1A families with putative nonrecurrent duplication. The duplications were identified by microsatellite typing and applying a CGH microarray. The breakpoint sequences in two families suggested an Alu-Alu-mediated rearrangement with the FoSTeS by the MMBIR, and a two-step rearrangement of the replication-based FoSTeS/MMBIR and meiosis-based recombination. The two-step mechanism has still not been reported. Segregation analysis of 17p12 microsatellite markers and breakpoint junction analysis suggested that the nonrecurrent rearrangements are stably inherited without alteration of junction sequence; however, they may allow some alteration of the genomic contents in duplication across generations by recombination event. It might be the first study on the pedigree analysis of the large CMT1A families with nonrecurrent rearrangements. It seems that the exact mechanism of the nonrecurrent rearrangements in the CMT1A may have a far more complex process than has been expected.

Association of a single nucleotide polymorphism in the 5' upstream region of the porcine myosin heavy chain 4 gene with meat quality traits in pigs.

We identified a potential molecular marker associated with meat quality traits in the myosin heavy chain 4, MYH4 gene of Landrace pigs. Sequencing revealed a single nucleotide polymorphism (SNP; g.-1398G>T) in the 5' upstream region of MYH4. It was significantly associated with the number of type IIa muscle fibers and water-holding capacity based on filter-paper fluid uptake. The GG genotype groups had a greater number of type IIa fibers and a larger area composed of type IIa fibers than the other genotype group (P = 0.004 and P = 0.061, respectively). Expression level of MYH4 gene in the genotype TT or GT was higher than in genotype of GG (P < 0.0001). The T allele may enhance expression level of MYH4 gene and then the portion of IIb type fiber in the muscle be increased by the T allelle. Therefore, we suggest that the g.-1398G>T in the 5' upstream region of the porcine MYH4 may be used as a molecular marker for meat quality traits, although its functional effect is not defined yet.

Whole-genome resequencing analyses of five pig breeds, including Korean wild and native, and three European origin breeds.

Pigs have been one of the most important sources of meat for humans, and their productivity has been substantially improved by recent strong selection. Here, we present whole-genome resequencing analyses of 55 pigs of five breeds representing Korean native pigs, wild boar and three European origin breeds. 1,673.1 Gb of sequence reads were mapped to the Swine reference assembly, covering ∼99.2% of the reference genome, at an average of ∼11.7-fold coverage. We detected 20,123,573 single-nucleotide polymorphisms (SNPs), of which 25.5% were novel. We extracted 35,458 of non-synonymous SNPs in 9,904 genes, which may contribute to traits of interest. The whole SNP sets were further used to access the population structures of the breeds, using multiple methodologies, including phylogenetic, similarity matrix, and population structure analysis. They showed clear population clusters with respect to each breed. Furthermore, we scanned the whole genomes to identify signatures of selection throughout the genome. The result revealed several promising loci that might underlie economically important traits in pigs, such as the CLDN1 and TWIST1 genes. These discoveries provide useful genomic information for further study of the discrete genetic mechanisms associated with economically important traits in pigs.

Acupuncture suppresses reinstatement of morphine-seeking behavior induced by a complex cue in rats.

Morphine causes physical and psychological dependence for individuals after repeated-use. Above all, our previous study showed that acupuncture attenuated reinstatement of morphine-seeking behavior induced by pharmacological cue. In this study, we investigated whether acupuncture could suppress the reinstatement of morphine-seeking behavior induced by the combination of environmental and pharmacological cues and the possible neuronal involvement. Male Sprague-Dawley rats were trained to self-administer morphine (1.0 mg/kg) for 3 weeks. Following the withdrawal phase (7 days), the effects of acupuncture on reinstatement of morphine-seeking behavior were investigated. For the investigation of neuronal involvement, the GABAA receptor antagonist bicuculline and the GABAB receptor antagonist SCH 50911 were pre-treated. Morphine-seeking behavior induced by combination of re-exposure to the operant chamber and morphine injection was suppressed perfectly by acupuncture at SI5, but not at the control acupoint LI5 and this effect was blocked by pre-treatment with the GABA receptor antagonists. This study suggests that acupuncture at SI5 can be considered as a predominant therapy for the reinstatement of morphine-seeking behavior in humans.

Increase in the Contents of Ginsenosides in Raw Ginseng Roots in Response to Exposure to 450 and 470 nm Light from Light-Emitting Diodes.

An light-emitting diode (LED)-based light source was used as a monochromatic light source to determine the responses of raw ginseng roots (Panax ginseng Meyer) to specific emission spectra with respect to the production of ginsenosides. The ginsenoside content in the ginseng roots changed in response to the LED light treatments at 25℃ relative to the levels in the control roots that were treated in the dark or at 4℃ for 7 d. Ginseng roots were exposed to LEDs with four different peak emission wavelengths, 380, 450, 470, and 660 nm, in closed compartments. Compared with the control 4℃-treated roots, roots that were treated with 450 and 470 nm light showed a significantly increased production of ginsenosides (p<0.05), with increases of 64.9% and 74.1%, respectively. The contents of the ginsenosides Rb2, Rc, and Rg1 were significantly higher (p<0.05) in the 450 and 470 nm-treated root samples. The ratio of protopanaxadiol ginsenosides (Rb1, Rb2, Rc, and Rd) to protopanaxatriol ginsenosides (Rg1, Rg2, Re, and Rf) was significantly higher (p<0.05) in the 450 and 470 nm-treated root samples than in the control 4℃-treated roots. This is the first report that demonstrates the increase and conversion of ginsenosides in raw ginseng roots in response to exposure to LED light.

Mutational analysis of whole mitochondrial DNA in patients with MELAS and MERRF diseases.

Mitochondrial diseases are clinically and genetically heterogeneous disorders, which make the exact diagnosis and classification difficult. The purpose of this study was to identify pathogenic mtDNA mutations in 61 Korean unrelated families (or isolated patients) with MELAS or MERRF. In particular, the mtDNA sequences were completely determined for 49 patients. From the mutational analysis of mtDNA obtained from blood, 5 confirmed pathogenic mutations were identified in 17 families, and 4 unreported pathogenically suspected mutations were identified in 4 families. The m.3243A>G in the tRNA(Leu(UUR))was predominantly observed in 10 MELAS families, and followed by m.8344A>G in the tRNA(Lys) of 4 MERRF families. Most pathogenic mutations showed heteroplasmy, and the rates were considerably different within the familial members. Patients with a higher rate of mutations showed a tendency of having more severe clinical phenotypes, but not in all cases. This study will be helpful for the molecular diagnosis of mitochondrial diseases, as well as establishment of mtDNA database in Koreans.